Hydrophobic surfaces, such as those of polystyrene, are nonspecifically active to the adsorption of various substances, such as biomolecules with hydrophobic portions, proteins, and the like. In an attempt to form specifically active surfaces, ligands which have specific reactivity have been covalently bonded to these surfaces. However, the covalent bonding to the surface is difficult to control, frequently resulting in splotchy coverage by the bonded ligand and several regions that are still nonspecific. Exemplary of methods used to immobilize a biomolecule upon a solid phase by covalent coupling are the coupling to agarose, crosslinked dextran or polyacrylamide or other hydrophilic polymers. Generally, functional groups on the solid substrate, such as hydroxide groups, are used to react with and covalently bind the molecule to be immobilized. A problem with these methods of immobilization is that they are not applicable to materials that lack such functional groups.
Another problem with covalently bound coatings, is that they, as a rule, are not removed from the substrate, which prevents recycling of the substrate. The porous polystyrene beads used in chromatography are expensive and after being coated are used for a specific separation and discarded when resolution is lost. It would be a significant cost savings if these beads could be stripped of the reactive coating and recycled by applying a new coating.
Certain biological molecules, such as enzymes and antibodies, can be immobilized by simple adsorption onto the solid phase. For example, an antibody may be adsorbed upon the surface of polystyrene in the form of a microliter plate, which is then used for heterogeneous enzyme immunoassay. The adsorbed biomolecule provides a specific enzymatic reaction or specific antibody-antigen interaction. Polystyrene is commonly used because of its transparency for colorimetric and photometric measurement, and because antibodies spontaneously bind to the polystyrene hydrophobic surface in a manner that usually preserves the immunochemical activity. However, it is necessary that essentially all of the hydrophobic sites on the surface be covered since they are non-specific adsorption sites. The background of the non-specific adsorption, if strong enough, can obscure the results of the assay. In addition, some biomolecules are difficult to adsorb upon polystyrene, and cannot be adsorbed to a sufficient extent for a practical assay. Others will only adsorb sufficiently under optimal conditions, which are often determined by trial and error. Further, some enzymes and antibodies lose activity when adsorbed upon a hydrophobic surface. For example, sensitive monoclonal antibodies sometimes lose activity due to conformational changes caused by the interaction with the hydrophobic surface. Therefore, the method of simple adsorption of active biomolecules upon a hydrophobic surface is not generally applicable.
It is known that certain block copolymers having a hydrophobic center block with hydrophilic end blocks can be used to coat hydrophobic surfaces. The center blocks are adsorbed onto the surface, with the end blocks extending from the surface and waving freely in a seaweed-like fashion. The coverage of the hydrophobic center blocks and the action of the end blocks effectively blocks the nonspecific adsorption sites and creates a nonadsorbing surface to certain substances such as proteins. A class of polymers commonly used in this application are the so-called Pluronic.TM. surfactants, which are triblock copolymers with the structure PEO--PPO--PEO (where "PEO" is poly(ethylene oxide) and "PPO" is poly(propylene oxide). Polymers of this type are also available under the name Poloxamer.TM.. In a specific application, Pluronic surfactants have been found to reduce the platelet adhesion and protein adsorption on surface treated glass or low density polyethylene. Pluronic surfactants have also been found to prevent bacterial adhesion on polystyrene surfaces. While a Pluronic coating essentially eliminates the non-specific reactivity of the substrate surface, the resulting hydrophilic surface has essentially no reactivity and is not a suitable surface for further adsorption of most biomolecules.
Many techniques for biochemical separation, such as low pressure affinity chromatography and immunological assays, are based on specific interactions between biomolecules of examination and chemical reagents immobilized on solid phase. However, because of the above difficulties encountered with covalently bound coatings and adsorbed coatings of reactive ligands, there is a need of a coating system that is generally applicable for different reactive ligands, has a higher degree of reactivity, and has little or no background non-specific reactivity.
Another aspect of biochemical separations are those based upon specific interaction between biomolecules to be examined and chemical reagents immobilized upon a solid phase. Such separations are the basis for, for example, low pressure affinity chromatography and solid phase immune assays.